PROJECT SUMMARY/ABSTRACT Despite the recent FDA approval of the oncolytic herpes simplex virus, ImlygicTM to treat melanoma, and the promising antitumor efficacy observed in many preclinical models using other similar viruses, strategies are still need to improve the anti-cancer effectiveness of herpes virotherapy. Our preliminarily results using a so-called ?directed evolution? strategy in a low-permissive cell culture support our overarching hypothesis that more effective virus mutants can be created via serial selection of mixed virus infections in poorly permissive cells. Combining sequencing analysis and CRISPR/Cas9 gene editing approaches, we developed our lead strain Mut-3?34.5, which is attenuated with RL1/?134.5 deletion. We found this novel virus induces a giant syncytia phenotype and contains five nonsynonymous alternations, including the A151T in Us8/gE, that potentially underlie the increased cancer cell killing effect of Mut-3?34.5. The aims of this project are: (1) Determine the mechanism(s) underlying the increased potency of Mut-3?34.5 virus in oncolysis, (2) Delineate the role of the non-parental non-synonymous mutations in Mut-3?34.5 and (3) Define the safety profile and efficacy of attenuated Mut-3?34.5 virus in animal models. Our overarching goal is to further develop more potent yet safe viral vectors for future clinical use.